The present invention relates to a pharmaceutical combination and its use in the treatment of disorders associated with excess benign and malignant cell proliferation, e.g. tumors or intimal cell proliferation.
There is a continuing need for the development of drugs having increased effectiveness in inhibiting or slowing down undesired cell proliferation, particularly in the cancer field and in vasculopathies.
Accordingly, there is provided a pharmaceutical combination comprising a compound of the somatostatin class, and a rapamycin macrolide.
The somatostatin class is a known class of small peptides comprising the naturally occurring somatostatin-14 and analogues having somatostatin related activity, e.g. as disclosed by A. S. Dutta in Small Peptides, Vol. 19, Elsevier (1993). By xe2x80x9csomatostatin analoguexe2x80x9d as used herein is meant any straight-chain or cyclic polypeptide having a structure based on that of the naturally occurring somatostatin-14 wherein one or more amino acid units have been omitted and/or replaced by one or more other amino radical(s) and/or wherein one or more functional groups have been replaced by one or more other functional groups and/or one or more groups have been replaced by one or several other isosteric groups. In general, the term covers all modified derivatives of the native somatostatin-14 which exhibit a somatostatin related activity, e.g. they bind to at least one somatostatin receptor (hSST-1, hSST-2, hSST-3, hSST4 or hSST-5), preferably in the nMolar range, more preferably to at least the hSST-2 receptor in the nMolar range.
Cyclic, bridge cyclic and straight-chain somatostatin analogues or derivatives are known and have been described together with processes for their production e.g. in U.S. Pat. Nos. 4,310,518 and 4,235,886, in European Patent Specifications EP-A-1295; 23,192; 29,310; 29,579; 30,920; 31,303; 63,308; 70,021; 83,305; 215,171; 203,031; 214,872; 143,307; 298,732; 277,419; 389,180; 395,417; 450,480A2; in Belgian Patent Specification BE-A-900,089; and in WO 91/09056; WO 97/01579; WO 97/14715, the contents thereof, in particular with respect to the compounds, being incorporated herein by reference.
Preferred somatostatin analogues are e. g. compounds of formula I 
wherein
A is C1-12alkyl, C7-10phenylalkyl or a group of formula RCOxe2x80x94, whereby
i) R is hydrogen, C1-11alkyl, phenyl or C7-10phenylalkyl, or
ii) RCOxe2x80x94 is
a) a D-phenylalanine residue optionally ring-substituted by halogen, NO2, NH2, OH, C1-3alkyl and/or C1-3alkoxy; or
b) the residue of a natural or a synthetic xcex1-amino-acid other than defined under a) above, or of a corresponding D-amino acid, or
c) a dipeptide residue in which the individual amino acid residues are the same or different and are selected from those defined under a) and/or b) above, the xcex1-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) being optionally mono- or di-C1-12alkylated or substituted by C1-8alkanoyl;
Axe2x80x2 is hydrogen or C1-3alkyl,
Y1 and Y2 represent together a direct bond or each of Y1 and Y2 is hydrogen
B is -Phe- optionally ring-substituted by halogen, NO2, NH2, OH, C1-3alkyl and /or C1-3alkoxy (including pentafluoroalanine), naphthylalanine or pyridylalanine,
C is (L)-Trp- or (D)-Trp- optionally xcex1-N-methylated and optionally benzene-ring-substituted by halogen, NO2, NH2, OH, C1-3alkyl and/or C1-3alkoxy,
D is Lys, 4-aminocyclohexylAla or 4-aminocyclohexylGly,
E is Thr, Ser, Val, Tyr, lie, Leu or an aminobutyric or aminoisobutyric acid residue,
G is a group of formula 
wherein
R7 is hydrogen or C1-3alkyl,
R10 is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, e.g. formnyl, C2-12alkylcarbonyl, benzoyl,
R11 is hydrogen, C1-3alkyl, phenyl or C7-10phenylalkyl
R12 is hydrogen, C1-3alkyl or a group of formula xe2x80x94CH(R13)xe2x80x94X1,
R13 is CH2OH, xe2x80x94(CH2)2xe2x80x94OH, xe2x80x94(CH2)3xe2x80x94OH, xe2x80x94CH(CH3)OH, isobutyl, butyl, benzyl, naphthyl-methyl or indol-3-yl-methyl, and
X1 is a group of formula 
wherein
R7 and R10 have the meanings given above,
R14 is hydrogen or C1-3alkyl,
R15 is hydrogen, C1-3alkyl, phenyl or C7-10phenylalkyl, and
R16 is hydrogen or hydroxy,
with the proviso that
when R12 is xe2x80x94CH(R13)xe2x80x94X1, then R11 is hydrogen or methyl,
wherein the residues B, D and E have the L-configuration, and the residues in the 2- and 7-position each independently have the (L)- or (D)-configuration,
in free form or in pharmaceutically acceptable salt or complex form.
Individual compounds of formula I suitable in accordance with the present invention are the following somatostatin analogues: 
A preferred compound of formula I is octreotide.
Compounds of formula I may exist e.g. in free form, salt form or in the form of complexes thereof. Acid addition salts may be formed with e.g. organic acids, polymeric acids and inorganic acids. Such acid addition salt forms include e.g. the hydrochlorides and acetates. Complexes are e.g. formed from compounds of the invention on addition of inorganic substances, e.g. inorganic salts or hydroxides such as Ca- and Zn-salts, and/or on addition of polymeric organic substances.
Further somatostatin analogues suitable for use in accordance with the present invention are:
cyclo [-Asn-Phe-Phe-DTrp-Lys-Thr-Phe-Gaba-], cyclo(Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser), and 
According to an alternatively preferred embodiment of the invention, the somatostatin component of the combination is a somatostatin analogue comprising the amino acid sequence of formula (II)
xe2x80x83xe2x80x94(D/L)Trp-Lys-X2xe2x80x94X3xe2x80x94xe2x80x83xe2x80x83(II)
wherein
X2 is a radical of formula (a) or (b) 
or 
wherein
R1 is optionally substituted phenyl,
R2 is xe2x80x94Z1xe2x80x94CH2xe2x80x94R1, xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94CH2xe2x80x94R1, 
wherein
Z1 is O or S, and
X3 is an xcex1-amino acid having an aromatic residue on the Cxcex1 side chain, or an amino acid unit selected from Dab, Dpr, Dpm, His,(Bzl)HyPro, thienyl-Ala, cyclohexyl-Ala and t.-butyl-Ala,
the residue Lys of said sequence corresponding to the residue Lys9 of the native somatostatin-14.
Such somatostatin analogues are e.g. disclosed in WO/ 97/01579, the contents thereof, in particular with respect to the specifically exemplified compounds, being incorporated herein by reference.
Preferably the sequence of formula II as defined above corresponds to the residues at positions 8 through 11 of the somatostatin-14. More preferably the somatostatin analogue as disclosed above comprises a hexapeptide unit, the residues at positions 3 through 6 of said hexapeptide unit comprising the sequence of formula II. More particularly the hexapeptide unit is cyclic, e.g. having a direct peptide linkage between the xcex1-carbonyl group of the residue at position 6 and the xcex1-amino group of the residue at position 1.
While Lys, X2 and X3 in the sequence of formula II have the L-configuration, Trp may have the D- or L-configuration, preferably the D-configuration.
X2 is preferably a residue of formula (a) or (b), R2 being preferably xe2x80x94Z1xe2x80x94CH2xe2x80x94R1 or 
When X3 comprises an aromatic residue on the Cxcex1 side chain, it may suitably be a natural or unnatural xcex1-amino acid, e.g. Phe, Tyr, Trp, Nal, Pal, benzothienyl-Ala, Tic and thyronin, preferably Phe or Nal, more preferably Phe. X3 is preferably an xcex1-amino acid bearing an aromatic residue on the Cxcex1 side chain.
When R1 is substituted phenyl, it may suitably be substituted by halogen, methyl, ethyl, methoxy or ethoxy e.g. in ortho and/or position. More preferably R1 is unsubstituted phenyl. Z1 is preferably O.
Representative somatostatin analogues comprising a residue of formula II are e.g compounds of formula (III) 
wherein
X2 and X3 are as defined above,
A1 is a divalent residue selected from Pro, 
xe2x80x83wherein R3 is NR8R9xe2x80x94C2-6alkylene, guanidino-C2-6alkylene or C2-6alkylene-COOH, R3a is H, C1-4alkyl or has independently one of the significances given for R3 R3bis H or C1-4alkyl, Ra is OH or NR5R6, Rb is xe2x80x94(CH2)1-3xe2x80x94 or xe2x80x94CH(CH3)xe2x80x94, R4 is H or CH3, R4a is optionally ring-substituted benzyl, each of R5 and R6 independently is H, C1-4alkyl, xcfx89-amino-C1-4alkylene, xcfx89-hydroxy-C1-4alkylene or acyl, R5ais a direct bond or C1-6alkylene, each of R8 and R9 independently is H, C1-4alkyl, xcfx89-hydroxy-C2-4alkylene, acyl or CH2OHxe2x80x94(CHOH)cxe2x80x94CH2xe2x80x94 wherein c is 0, 1, 2, 3 or 4, or R8 and R9 form together with the nitrogen atom to which they are attached a heterocyclic group which may comprise a further heteroatom, and R17 is optionally ring-substituted benzyl, xe2x80x94(CH2)1-3xe2x80x94OH, CH3xe2x80x94CH(OH)xe2x80x94 or xe2x80x94(CH2)1-5xe2x80x94NR5R6, and
ZZa is a natural or unnatural xcex1-amino acid unit.
ZZa may have the D- or L-configuration. When ZZa is a natural or unnatural xcex1-amino acid unit, it may suitably be e.g. Thr, Ser, Ala, Val, Ile, Leu, Nle, His, Arg, Lys, Nal, Pal, Tyr, Trp, optionally ring-substituted Phe or Nxcex1-benzyl-Gly. When ZZa is Phe, the benzene ring thereof may be substituted by e.g. NH2, NO2, CH3, OCH3 or halogen, preferably in para position. When ZZa is Phe, the benzene ring thereof is preferably unsubstituted.
When A1 comprises a Pro amino acid residue, any substituent present on the proline ring, e.g. R3xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94 etc., is preferably in position 4. Such substituted proline residue may exist in the cis form, e.g. 
as well as in the trans form. The present invention covers each geometric isomer individually as well as mixtures thereof.
When A1 is (NR8R9xe2x80x94C6-2alkylene-NHxe2x80x94COxe2x80x94)Pro- where NR8R9 forms a heterocyclic group, such group may be aromatic or saturated and may comprise one nitrogen or one nitrogen and a second heteroatom selected from nitrogen and oxygen. Preferably the heterocyclic group is e.g. pyridyl or morpholino. C2-6Alkylene in this residue is preferably xe2x80x94CH2xe2x80x94CH2xe2x80x94.
Any acyl as R5, R6, R8 and R9 in A1 may be e.g. R18COxe2x80x94 wherein R18 is H, C1-4alkyl, C2-4alkenyl, C3-6cycloalkyl or benzyl, preferably methyl or ethyl. When R4a, or R17 in A1 is ring-substituted benzyl, the benzene ring may be substituted as indicated above for ZZa.
A preferred group of compounds of formula III are such wherein A1 is free of a lateral xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94 moiety. A further group of preferred compounds of formula III are such wherein A1 comprises a basic lateral radical, e.g. a R3xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94 or 
moiety.
A still further group of preferred compounds of formula III are such wherein the N-terminal amino acid comprises a substituted Pro, particularly 4-substituted Pro, e.g. compounds of formula III wherein A1 is 4-substituted Pro.
Preferably A1 is 4-(R3xe2x80x94NHxe2x80x94COxe2x80x94O)Pro.
Examples of somatostatin analogues comprising a residue of formula II include e.g. cyclo [4xe2x80x94(NH2xe2x80x94C2H4xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94)Pro-Phe-DTrp-Lys-Ser(Benzyl)-Phe].
The term xe2x80x9cmacrolidexe2x80x9d as used herein, refers to a macrocyclic lactone, for example a compound having a 12-membered or larger lactone ring. Of particular interest are the xe2x80x9clactam macrolidesxe2x80x9d, i.e. macrocyclic compounds having a lactam (amide) bond in the macrocycle in addition to a lactone (ester) bond, for example rapamycin and its numerous derivatives and analogues. Rapamycin is an immunosuppressive lactam macrolide that is produced by Streptomyces hygroscopicus, and having the structure depicted in Formula 
See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat. No. 3 929 992. One group of rapamycin derivatives are 40-0-substituted derivatives of rapamycin having the structure of Formula IV: 
wherein
X4 is (H,H) or O;
Y3 is (H,OH) or O;
R20 and R21 are independently selected from H, alkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkoxycarbonylalkyl, hydroxyalkylaryalkyl, dihydroxyalkylarylalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl, dioxolanylallyl, dialkyl-dioxolanylalkyl, di(alkoxycarbonyl)-triazolyl-alkyl and hydroxyalkoxy-alkyl; wherein xe2x80x9calk-xe2x80x9d or xe2x80x9calkylxe2x80x9d refers to C1-6alkyl, branched or linear, preferably C1-3alkyl,; xe2x80x9carylxe2x80x9d is phenyl or tolyl; and acyl is a radical derived from a carboxylic acid; and
R22 is methyl or R22 and R20 together form C2-6alkyl; provided that R20 and R21 are not both H; and hydroxyalkoxyalkyl is other than hydroxyalkoxymethyl.
Such compounds are disclosed in WO 94/09010 the contents of which, in particular with respect to the specifically exemplified compounds, are incorporated herein by reference.
A preferred compound is e.g. 40-O-(2-hydroxy)ethyl-rapamycin (referred thereafter as Compound B).
Further preferred rapamycin derivatives are e.g. those disclosed in WO 96/41807, the contents thereof, in particular with respect to the specifically exemplified compounds of formula I disclosed therein, being incorporated herein by reference. Particularly preferred are 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxyethyl)-rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-rapamycin and 1 6-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.
Further rapamycin derivatives are known, e.g. carboxylic acid esters such as disclosed in WO 92/05179, amide esters such as disclosed in U.S. Pat. No. 5,118,677, carbamates such as described in U.S. Pat. No. 5,118,678, fluorinated esters such as disclosed in U.S. Pat. No. 5,100,883, acetals, e.g. in U.S. Pat. No. 5,151,413, silyl ethers, e.g. in U.S. Pat. No. 5,120,842, arylsulfonates and sulfamates, e.g. in U.S. Pat. No. 5 177 203, derivatives wherein the methoxy group at the position 16 is replaced with alkynyloxy, e.g. in WO 95/16691 and further derivatives such as disclosed in WO 93/11130, WO 94/02136, WO 94/02385 and WO 95/14023, all incorporated herein by reference.
Rapamycin and above mentioned derivatives have been shown to have potent immunosuppressant properties. Rapamycin has also been shown to inhibit smooth muscle cell proliferation and to inhibit cancer growth.
Somatostatin analogues, e.g. octreotide, vapreotide and lanreotide, have been disclosed i.a. to inhibit growth hormone secretion and to have an inhibiting effect on malignant tumor growth, e.g. in breast cancer. Octreotide and lanreotide have also been disclosed to inhibit smooth muscle cell proliferation.
In accordance with the invention, it has now surprisingly been found that a combination of 2 active ingredients believed to act on basically different mechanisms such as a somatostatin analogue and rapamycin or a derivative thereof, can be combined and synergistically inhibit cell hyperproliferation.
In accordance with the particular findings of the present invention, there is provided in a first aspect:
1. Use of a compound of the somatostatin class, in free form or in pharmaceutically acceptable salt form, for manufacturing a pharmaceutical composition for use in synergistically effective amounts in the prevention or treatment of cell hyperproliferation in combination with a rapamycin macrolide, e.g. for the manufacture of a kit as disclosed hereinafter.
2. Use of a compound of the somatostatin class, in free form or in pharmaceutically acceptable salt form, in combination in synergistically effective amounts with a rapamycin macrolide for the prevention or treatment of cell hyperproliferation.
3. A method for preventing or treating cell hyperproliferation in a subject in need of such treatment which comprises administering to such subject a synergistically effective amount of a compound of the somatostatin class in free form or in pharmaceutically acceptable salt form, and a rapamycin macrolide.
4. A kit or package for the treatment or prevention of cell hyperproliferation, said kit or package including a pharmaceutical composition comprising a compound of the somatostatin class in free form or in pharmaceutically acceptable salt form, and a pharmaceutical composition comprising a rapamycin macrolide. The kit or package may also contain instructions to use the pharmaceutical compositions in accordance with the present invention.
According to the invention, the combination of a compound of the somatostatin class and a rapamycin macrolide is indicated for the prevention or treatment of malignant tumor growth, e.g. breast, lung, GEP tumors, pituitary adenomas, lymphomas, etc., for the prevention or treatment of proliferative vascular diseases, e.g. biologically or mechanically induced vascular injury causing intimal thickening, e.g. restenosis, atherosclerosis, vascular occlusion, injury following percutaneous transluminal coronary angioplasty, vascular surgery or transplantation surgery, transplant vasculopathies, for example chronic rejection of various tissues and organs such as heart, kidney, pancreas, lung, liver, bowel, trachea and combined heart-lung.
The combination is particularly indicated for preventing intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion in a mammal.